The present invention arises from the discovery that clearance of human tissue-type plasminogen activator from the circulation can be manipulated by altering the molecule's carbohydrate structure.
Tissue-type plasminogen activator (t-PA) is a serine protease that is involved in fibrinolysis. Binding of t-PA to a fibrin clot causes an enhancement of plasminogen activation (Hoylaerts, M. et al., J. Biol. Chem. 257:2912-2919 [1982]; Rijken, D. C., et al., J. Biol. Chem. 257:2920-2925 [1982]). Human tissue-type plasminogen activator converts plasminogen to plasmin. The plasmin, so produced, proteolytically cleaves fibrin matrices which comprise the backbone of blood clots. Human tissue-type plasminogen activator thereby mediates the dissolution of blood clots and is consequently useful in the treatment of various thrombolytic disorders. Although t-PA has been isolated from many sources such as the Bowes melanoma cell line (Rijken, D. C. et al., J. Biol. Chem. 256:7035-7041 [1981]; Wallen, P., et al., Eur. J. Biochem. 132:681-686 [1983]), uterine tissue (Rijken, D. C. et al. Biochim. Biophys. Acta 580:140-153 [1979]) and blood vessel perfusates (Binder, B. R., et al., J. Biol. Chem. 254:1998-2003 [1979]), the advent of recombinant DNA technology has allowed the production of t-PA (Pennica, D. et al., Nature (London) 301:214-221 [1983]) in sufficient quantities to perform clinical trials on patients with myocardial infarction, peripheral vascular thrombi and pulmonary embolism. These trials have shown that recombinant t-PA (rt-PA) is an extremely effective thrombolytic agent with minimal effects on fibrinogen levels in the blood (Williams, D. O., et al., Circulation 73:338-346 [1986]; Graor, R. A. et al., Circulation 74(suppl.1):1-15-1-20 [1986]; Collen, D., et al., Circulation 73:511-517 [1986]).
The elimination of rt-PA from the circulation is relatively rapid. The biphasic clearance is dominated by an alpha phase with a half life of about 2 min in rabbits (Korninger, C., et al., Thromb. Haemos. 46:658-661 [1985]; Nilsson, S., et al., Thromb. Res. 39:511-521; Bounameaux, H., et al., Blood 67:1493-1497 [1986]) and 4 min (Baughman, R. A., Tissue Plasminogen Activator in Thrombolytic Therapy (Sobel, et al., eds.) pp. 41-53, Marcel Dekker, N.Y. [1987]) to 6 min (Wallen, P. et al., supra) in humans. The liver is the most significant site of t-PA uptake and metabolism (Korninger, C., et al., supra; Nilsson, S., et al., supra; Bounameaux, H., et al., supra). A number of receptor systems in the liver have been described which recognize specific terminal residues on the oligosaccharide portions of glycoproteins. Proteins cleared by this mechanism also have half lives on the order of a few minutes (Ashwell, G. et al., Ann. Rev. Biochem. 51:531-554 [1982]).
Recombinant t-PA has four potential sites for N-linked glycosylation. A high mannose oligosaccharide is present at position 117 and a complex oligosaccharide at position 448. At position 184 a complex oligosaccharide is present in type I rt-PA and absent in type II rt-PA; the ratio of type I to type II is about 1 to 1. A fourth potential glycosylation site at residue 218 is not glycosylated. The glycosylation pattern of rt-PA is similar to that of melanoma-derived t-PA.
The abbreviation t-PA for human tissue-type plasminogen activator was adopted after proposal at the XXVIII Meeting of the International Committee on Thrombosis and Hemostatis, Bergamo, Italy, 27 Jul. 1982. As used herein, the terms "human tissue-type plasminogen activator", "t-PA", "human t-PA" or "tissue plasminogen activator" denote human extrinsic (tissue-type) plasminogen activator, produced, for example, from natural source extraction and purification [see Collen et al., European Patent Application No. 41766 (published 16 Dec. 1981 based upon a first filing of 11 Jun. 1980) and Rijken et al., Journal of Biol Chem. 256, 7035 (1981), incorporated herein by reference, and by recombinant cell culture systems as described together with its amino acid sequence and physical and biological characteristics, for example, in European Patent Application Publication No. 93619, (published 9 Nov. 1983) based upon a first filing of 5 May 1982), incorporated herein by reference.
U.S. Pat. No. 4326033 reports extending the half-life of urokinase by chemically modifying its carbohydrate structure. Urokinase is immunologically distinct from human tissue-type plasminogen activator. There is no justification, either from U.S. Pat. No. 4,326,033 or otherwise, in considering that such carbohydrate modifications of urokinase would have applicability to other glycoproteins. Indeed, for example, removal of sialic acid from ceruloplasmin decreases its half-life dramatically; yet, identical treatment of transferrin, another serum glycoprotein, has no significant effect on half-life. (Sharon, Complex Carbohydrates, Addison-Wesley Publ. Co., p. 194-196, (1975); see also Ashwell et al., Adv. Enzymology 41, 99 (1974) and Alexander et al., Science 226, 1328 (1984).
The elimination of melanoma-derived t-PA in mice has been reported to be unaffected by titration of the active site with phenylmethylsulfonyl fluoride or by the coinjection of diisopropylfluorophosphate thrombin, asialoorosomucoid, macroalbumin or excess unlabeled t-PA (Fuchs, H. E., et al. Blood 65:539-544 [1985]). The results obtained with asailoorosomucoid and macroalbumin would indicate that carbohydrate structures do not influence the clearance of t-PA. The clearance of melanoma derived t-PA has been reported to be unaffected by monosaccharides in rats (Emeis, C. M., et al., Thromb. Haemos. 54:661-664 [1985]). On the other hand, when the cellular components of the rat liver were separated, the endothelial cells were reported to take up melanoma t-PA by a pathway that could be inhibited by high mannose glycoproteins; this result was interpreted to mean that t-PA is taken up into the liver at least in part by the mannose receptor (Einarsson, M., et al., Thromb. Haemos. 54:270 [1985]). In other in vitro studies, rt-PA was shown to bind with high affinity to rat liver hepatocytes. The receptor did not appear to be carbohydrate-dependent (Bakhit, C., et al., J. Biol. Chem, 262:8716-8720 [1987]).
In Patent Application International Publication No. WO84/01786, published 10 May 1984 based upon a first filing of 28 Oct. 1982, there is described an indiscriminate modification of tissue-type plasminogen activator resulting in a molecule with reduced biological activity and purported increased half-life, compared with the unmodified polypeptide. The single example involves treating a partially purified human tissue-type plasminogen activator with sodium periodate giving a product reported to have about 70 to 90 percent of the original (unmodified) activity. There is no indication in WO84/01786 of an appreciation of the nature and characterization of the carbohydrate structures present in native material, and more importantly, in the modified form they produce. In fact, periodate is known to modify or disrupt, by oxidation, all carbohydrate structures without their concomitant, substantial removal from amino acid linkage.
There is also no indication in WO84/01786 of how many such structures human tissue-type plasminogen activator has, or what the actual carbohydrate make-up is, either for unmodified or their modified version. Thus, their periodate treated molecule was most probably modified by oxidation of all carbohydrate structures indiscriminately, without focus on a particular site.
Recently, it was reported that the in vivo clearance rates of glycosylated and deglycosylated human tissue-type plasminogen activator were not significantly different, forcing the conclusion that the clearance rate of human tissue-type plasminogen activator is not affected by the absence of carbohydrates (Larsen et al., Proteases in Biological Control and Biotechnology, UCLA Symposium Park City, Utah,. Feb. 9-14, 1986. See Little et al., Biochemistry 23, 6191 (1984).
It is an object of the present invention to provide novel t-PA variants by modifying its carbohydrate structure. Another object of this invention is to provide t-PA variants that can be cleared from the circulation at different rates. Yet another object of this invention is to provide effective thrombolytic agents suitable for treatment of diverse clinical conditions.